Alcohol use disorders are common and incur a huge cost to society. Unfortunately, pharmacotherapy of alcohol use disorders is restricted to three FDA approved drugs: disulfuram, naltrexone, and acamprosate. Therefore, there is considerable need for the development of new agents to treat these disorders. Mounting evidence suggests that protein kinase C epsilon (PKC) is a good target for development of drugs to treat alcohol use disorders, based mainly on studies using gene-targeted mice that lack this enzyme (Prkce-/- mice). These mice drink substantially less ethanol than wild type mice, show heightened aversion to ethanol, are more easily intoxicated by low doses of ethanol, and recover from intoxication very slowly compared with wild type mice. These behaviors are associated with enhanced function of GABAA receptors, which are the major mediators of inhibitory neurotransmission in the nervous system. PKC regulates GABAA receptors by phosphorylating GABAA2 subunits at Ser-327, which reduces their sensitivity to benzodiazepines and ethanol. PKC also phosphorylates the N-ethylmaleimide sensitive factor (NSF), at Ser-460 and Thr-461, which stimulates removal of GABAA receptors from synapses. The long-term goal of this project is to identify targets for the development of new drugs to treat alcohol use disorders. The objective of this application is to specifically validate PKC as a drug target and identify additional drug targets within neuronal PKC signaling pathways. The main hypothesis to be tested is that selective inhibitors of PKC signaling can reduce voluntary ethanol consumption in mice. Three aims are proposed. The first aim will determine the consequences of administering a highly selective PKC inhibitor and validate PKC as a drug target to decrease alcohol consumption. This will be achieved by using a new line of mutant mice in which the ATP binding pocket of PKC has been modified to allow highly specific inhibition by small molecules that do not inhibit native kinases. In the second aim, three novel, related compounds that are potent inhibitors of PKC will be administered to wild type mice to determine if this class of compounds should be developed further as drugs to treat alcohol use disorders. The third aim will use a newly developed proteomics approach to identify direct substrates of PKC for study of their role in regulating GABAA receptors and behavioral responses to ethanol. Because of its focus on identifying new drug targets and pharmacological agents to treat alcohol use disorders, this project has high translational value for serving public health.